Otitis media (OM) is the most common reason that an ill child sees a health care provider, receives an antimicrobial or undergoes a general anesthetic. The overprescribing of antibiotics to treat OM is one of the major causes of antibiotic-resistant bacteria. Previous work from the Center for Genomic Sciences (CGS) has shown that culturally-negative middle-ear effusions actually contain bacterial DNA, mRNA and protein. These findings led to the rejection of the previous dogma, which held that chronic OM was a non-bacterial, inflammatory process, with the paradigm that OM is a mucosal biofilm disease. In conjunction with the Center for Biofilm Engineering, CGS has demonstrated that Hemophilus influenza can form biofilms in an animal model and in pediatric otorrhea. Biofilms are complex organizaton of bacteria covered with a protective exopolysaccharide matrix. In this state, bacteria are slow-growing and very resistant to antibiotics. In this application, we extend our work in biofilms by investigating Streptococcus pneumoniae, one of the most common bacteria associated with OM, and the most common respiratory pathogen. Our hypothesis is that understanding the differences in biofilm and planktonic gene expression will identify proteins that can serve either as immunogens for vaccine development, or targets for novel antimicrobial agents. Using state-of- the-art microarray technologies and a robust animal model of OM, the chinchilla, we propose to identify those genes that are necessary for S. pneumoniae to form a biofilm. Combining advances in imaging and molecular biology, we will directly examine the role of the differentially-regulated genes identified by the array technology. Delineating the molecular basis of biofilms will not only advance our understanding of OM, but of chronic infectious disease in general.